Resonance Energy Transfer

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Resonance Energy Transfer
Non-Radiative Energy Transfer

• Fluorescence Resonance Energy Transfer
• Surface Energy Transfer

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• - Multiplicity A property of a system due to
  the spin, or angular
• momentum, of its component
  particles ( e.g., electrons)
• Number of states with a given angular momentum
• 2 S 1, S total spin
• if S0 singlet
• if S 1/2 doublet
• if S 1 triplet

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Fluorescence
One of a class of luminescence phenomena in
which certain molecules may emit light with a
longer wavelength than the light with which were
excited
k f
D hv E
D
D hv F
k i
D
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• Quantum yield Q
• The ratio of the number of photons emitted
  to the number of photons absorbed

• Lifetime
• The average time spent in the excited state
  before returning to the ground state

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FRET Fluorescence resonance energy transfer

• A distance dependent physical process by
  which energy is transferred nonradiatively from
  an excited molecular fluorophore (Donor) to
  another fluorophore (Acceptor) by means of
  intermolecular long-range dipoledipole coupling

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Energy Level Diagram
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The reaction scheme
D- donor A- accepter hv e,d,f - photon energy
by each frequencies K di,ai - radiatuibless
decay constants K d - radiative decay rate of
the donor in the absence of accepter K a -
radiative decay rate of the accepter K t - rate
of energy transfer
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Donor quantum yield Q
The donor quantum yield Q in the presence of
acceptor (Qda) and in the absence of acceptor
(Qd)
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FRET Efficiency E
By measuring the fluorescence intencities of the
donor with acceptor (Qda) and without acceptor
(Qd).
By using the lifetime of the donor in presence
(Tda) and absence of the acceptor (Td)
where
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Förster distance
The relationship between the transfer efficiency
and the distance between the two probe (R)
Ro the Förster distance at which the energy
transfer is (on average) 50
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Ro can be calculated using
Qd the quantum yield of the donor, n the
refractive index of the medium (generally
assumed to be 1.4 for proteins) Nav
Avogadro's number (Nav 6.02 x 10 per
mole) Kappa squared the orientation factor J
the overlap integral
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Kappa squared
Orientation factor , kappa squared
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The overlap intergral J
The degree of overlap between the donor
fluorescence spectrum and the acceptor absorption
spectrum
? the wavelength of the light e(?) the
molar extinction coefficient of the acceptor at
that wavelength f the fluorescence spectrum of
the donor normalized on the wavelength scale
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Surface Energy Transfer

• Energy transfer from a dipole to a metallic
  surface
• Interaction of the electromagnetic field of the
  donor dipole with the nearly free conduction
  electrons of the accepting metal
• Surface energy transfer efficiency
• KSET (1/tD) ( do/d)4
  

Yun et al., JACS, 2005, 127, 3115-3119
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• Schematic representation of the system we
  are studying, which consists of a fluorescein
  moiety (FAM) appended to ds-DNA of length R
  (varying from 15 to 60bp) with a Au nanoparticle
  (d 1.4 nm) appended to the other end. The
  flexible C6 linker produces a cone of uncertainty
  ( R) for both moieties. Addition of M.EcoRI
  (methyltransferase) bends the ds-DNA at the
  GAATTC site by 128 , producing a new effective
  distance R'.

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• Energy transfer efficiency plotted versus
  separation distance between FAM and Au(NM).
  Filled circles () represent DNA lengths of 15bp,
  20bp, 30bp, and 60bp. The measured efficiencies
  of these strands with the addition of M.EcoRI are
  represented by the open circles ( ). The error
  bars reflect the standard error in repeated
  measurements of the fluorescence as well as the
  systematic error related to the flexibility of
  the C6 linker as illustrated in Figure 1. The
  dashed line is the theoretical FRET efficiency,
  while the solid line is the theoretical SET
  efficiency

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• Conditions
• -Overlapping of Donar emission and Acceptor
  Excitation spectrum.
• -FRET Donor/Acceptor lt10nm.
• -SET Donor/Metal lt20 nm
• -Spectrally distinct
• Applications
• -Biomolecular interaction study in vivo/vitro
• -In vivo imaging/co-localization study
• - Biosensing
• Pairs (http//probes.invitrogen.com/resources/
  //microscopy.biorad.com)
• - Organic dye
• -ALEA-488/RHOD-2 FITC/RHOD-2 FITC/TRITC
  GFP/RHOD-2
• - Fluorescent protein
• -BFP/GFP BFP/YFP BFP/RFP CFP/YFP
• - Nanocrystal
• -QD/QD QD/gold

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• Examples of available fluorescent dye and
  quencher families, almost all of which have been
  used for FRET measurements. Absorbance and
  emission maxima along with spectral regions
  covered by a particular dye family are
  highlighted. Tetramethylrhodamine (TMR),
  carboxytetramethylrhodamine (TAMRA), and
  carboxy-X-rhodamine (ROX) are all rhodamine-based
  dyes. The most common D/A dye combinations are
  coumarin/fluorescein, fluorescein/rhodamine, and
  Cy3.5/Cy5. Popular dye/quencher combinations
  include rhodamine/Dabcyl and Cy3/QSY9. Major
  suppliers are the companies Molecular Probes
  (fluorescein, rhodamine, AlexaFluor, BODIPY
  Oregon Green, Texas Red, and QSY quenchers),
  Amersham Biosciences (Cy dyes and Cy5Q/Cy7Q
  quenchers), AnaSpec (HiLyte Fluors, QXL
  quenchers), ATTO-TEC (ATTO dyes and quenchers),
  and Molecular Biotechnology (DY dyes), Pierce
  (DyLight 547 and DyLight 647 dyes), Berry and
  Associates (BlackBerry), and Biosearch
  Technologies (Black Hole). FITCfluorescein
  isothiocyanate.

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Experimental methods
Conventional filter FRET
Apply filter/emission band configurations for
donor, acceptor and FRET (donor excitation and
acceptor emission) to acquire single images or
time series.
If the donor signal decreases, acceptor and
FRET signal increases.
Acceptor photobleaching
Apply donor/ acceptor configurations to acquire
single images or time series. After some control
images, acceptor (with 514 nm) is bleached.
Donor signal increases after acceptor bleach.
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Analysis of FRET

• Fluorescence lifetime imaging microscopy (FLIM)
• Information about the interactions between, and
  the structural
• states of, signaling molecules needs to be
  obtained as a
• function of space and time in a living cell.
• By using FLIM, the nanosecond decay kinetics of
  the electronic
• excited-state of fluorophores can be mapped
  spatially.
• Fluorescence lifetime
• The average amount of time that a molecule spends
  in the
• excited state upon absorption of a photon of
  light.
• Fluorescence lifetime is independent of
  fluorophore
• concentration and light-path length.

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Time domain FLIM
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Fluorephore materials used in bioanalytical FRET

• Organic materials
• - Available in reactive form fromcommercial
  sources activated with N- hydroxysuccinimide
  (NHS) ester, maleimide, hydrazide, amine
  functionality
• - Ex) Fluorescein dyes very popular
  because of their high quantum yield, solubility,
  ease of bioconjugation. Excitation with a
  standard argon-ion laser (488 nm)
• High rate of photo-bleaching, pH
  sensitive, self-quenching
• - Alternatives AlexaFluore, Cy family,
  BODIPY
• Inorganic materials
• - Metal chelates, semiconductor nanocrystals
• Biological origins
• - Fluorescent proteins

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• Structures of common UV/Vis fluorescent
  dyes. Typical substituents at the R position
  include CO2-, SO3-, OH, OCH3, CH3, and NO2 Rx
  marks the typical position of the bioconjugation
  linker.

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Photochromic dyes

• - Photochromic compounds having the ability
  to undergo reversible transformation in response
  to illumination at appropriate wavelengths
• - High local sensor densities, irreversible
  photo-bleaching with continuous monitoring

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• a) Structure of sulfo-NHS-BIPS (sulfo-NHSN-hydrox
  ysulfosuccinimide sodium salt BIPS1 ,3 ,3
  -trimethylspiro2H-1-benzopyran-2,2 -indoline)
  in the spiropyran (SP) form before (left) and
  merocyanine (MC) form after (right) conjugation
  to a protein. b) Schematic representation of
  quantum dot (QD) modulation by photochromic FRET
  after interacting with MBP-BIPS
  (MBPmaltose-binding protein). When BIPS is
  converted to the MC form by UV light, the QD
  emission is reduced through FRET quenching. After
  photoconversion with white light to the SP form,
  the direct emission of the QD is substantially
  increased. c) Photoluminescence spectra of the
  555-nm luminescing QD 20 MBP-BIPS system with a
  dye/protein ratio of 5 after photoconversion from
  the SP to the MC form. d) Effect of pcFRET on QD
  photoluminescence (initial change from white
  light to UV). Figure adapted from reference 106
  with permission of the American Chemical Society.

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Fluorescent proteins

• Green Fluorescence Protein (GFP) from jellyfish
• Widespread use by their expression in other
  organisms
• Key internal residues are modified during
  maturation to form
• the p-hydroxybenzylideneimidazolinon
  chromophore, located in the central helix and
  surrounded by 11 ß-strands (ß-can structure)
• In-vivo labeling of cells Localization and
  tracing of target protein
• GFP variants
• - BFP, CFP, YFP
• Red fluorescent protein (DS Red) from coral reef
  tetrameric, slow maturation
• Monomeric RFP by protein engineering
• Quantum yield 0.17 (BFP) 0.79 (GFP)
• BFP/CFP CFP/YFP( high change in the FRET signal
  ratio)
• usually fused to N- or C terminus of
  proteins by gene manipulation

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GFP (Green Fluorescent Protein)

• Jellyfish Aequorea victoria
• A tightly packed ?-can (11 ?-sheets) enclosing an
  ?-helix containing the chromophore
• 238 amino acids
• Chromophore
• Cyclic tripeptide derived from Ser-Tyr-Gly
• The wt GFP absorbs UV and blue light (395nm and
  470nm) and emits green light (maximally at 509nm)

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• a) Normalized absorption and b)
  fluorescence profiles of representative
  fluorescent proteins cyan fluorescent protein
  (cyan), GFP, Zs Green, yellow fluorescent protein
  (YFP), and three variants of red fluorescent
  protein (DS Red2, AS Red2, HC Red). From
  Clontech.

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Use of Fluorescent proteins for investigation of
biomolecular interactions
Inter-molecular FRET
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FRET-based Sensors
Intra-molecular FRET
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Calmodulin

• Calcium ions play a crucial role in the
  metabolism and physiology of eukaryotes
• Cells have developed a multitude of ways to
  control and make use of this ion gradient to
  regulate many cellular processes, ranging from
  transcription control and cell survival to
  neurotransmitter release and muscle function.
• Calmodulin (CaM, 148 aa) is a ubiquitous,
  calcium-binding protein ( typically binds 0, 2 or
  4 Ca2) that can bind to and regulate a multitude
  of different protein targets, thereby affecting
  many different cellular functions.
• CaM mediates processes such as inflammation,
  metabolism, apoptosis, muscle contraction,
  intracellular movement, short-term and long-term
  memory, nerve growth and the immune response.
• In the absence of Ca2, the two main helical
  domains have hydrophobic cores. On the binding of
  a calcium ion, conformational changes, which are
  mediated by non-covalent interactions, expose
  hydrophobic regions which have the potential to
  act as docking regions for target proteins (
  over 100 proteins including kinases, phosphatases
  etc.)

In the absence of Ca2
In the presence of Ca2
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• Modified MBP fluorescent indicator. ECFP
  as donor was fused to the N terminus of MBP, and
  YFP as a FRET acceptor was fused to the C
  terminus. H indicates the portion of protein
  functioning as a hinge between the two lobes of
  the MBP. The central binding pocket of the MBP is
  located between the two lobes. In the absence of
  maltose, the two FPs are at their maximum
  distance from each other and FRET is minimal.
  Upon binding maltose, the MBP undergoes a
  conformation change that brings the two FPs into
  close proximity and increases FRET, which can be
  monitored by the change in ratio of the YFP and
  CFP emission

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• a) Confocal image of a maltose-FP sensor
  expressed in yeast. Fluorescence is detected in
  the cytosol but not in the vacuole. Scale bar1
  um.
• b) Changes of the maltose concentration in
  the cytosol of yeast that expresses a maltose
  sensor with a Kd value of 25 M. The graph
  indicates emission ratio as a function of maltose
  uptake for a single yeast cell.

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Activation of protein kinase Ca using FLIM
Science, 283, 2085-2089, 1999
Activation of GFP-tagged protein kinase C a (PKC
a) in live Cos7 cells
(a) Fluorescence images of GFP-PKCa (b)
Fluorescence lifetime images of GFP-PKC a within
the middle microinjected cell owing to FRET
between GFP-PKCa and site-specific IgG-Cy5
Binding of Antibody to phosphorylated PKCa that
is induced by TPA TPA tetradecanoyl
phorbolacetate
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Enzyme-generated Bioluminescence

• BRET ( Bioluminescence RET)
• - Donor Luciferase Acceptor GFP
• - No excitation light source to excite the
  donor, which avoids problems such as light
  scattering, high background noise, and direct
  acceptor excitation
• In-vivo monitoring of protein-protein
  interactions such as circadian clock proteins,
  insulin receptor activity, real-time monitoring
  of intracellular ubiquitination
• The firefly luciferase/luciferin system the
  best candidate for a BRET-based donor high
  quantum yield ( 0.88)

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• Bioluminescent substrates and enzymatic
  reactions of several common luciferases
• a) the aliphatic aldehyde substrate of
  bacterial luciferase
• b) structure and reaction of luciferin,
  the substrate of firefly luciferase
• c) colenterazine, the substrate for
  Renilla luciferase and also part of
• apoaequorin.

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Enzyme-generated Chemiluminescence
Luminophore is a synthetic substrate that is
excited through an enzymatically catalyzed
reactions

• Chemiluminescent substrates and the
  enzymatic reactions of horseradish peroxidase
  (HRP) and alkaline phosphatase. a) Luminol b)
  Acridan (also available as an ester) c)
  Adamantyl-1,2-dioxetane (substrate for alkaline
  phosphatase and other enzymes).

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Gold nanoparticles

• Exceptional quenching ability
• Plasmon resonances in the visible range with
  large extinction coefficient (105 /cm/M)
• Stable
• Unfluctuating signal intensities
• Resistant to photo-bleaching

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Gold Nano Particles (AuNPs)

• Core Materials for NPs
• - Au, Ag, Pt Electron transporter,
  Catalysis, NPs coating for electrode
• - Mg, Co, Fe Magnetic behavior,
  Sample purification, MRI signal enhancing
• - CdSe, ZnS, InP Semiconductor QDs
• Stabilization by surfactants in synthesis of
  AuNPs
• - Reduction of HAuCl4 in the presence of
  surfactant
• - Citrate, tannic acid, white phosphorus
  gt 3 nm
• - Alkanethiol Monolayer protected
  cluster (MPC), 2 3 nm
• - Dendrimer Dendrimer encapsulated
  nanocluster (DEN). lt 2 nm
• Characteristics of AuNPs
• - Surface Plasmon Resonance Band
• . Absorbance band near 520 nm in
  5 several tens nm of AuNPs
• . SPB shift responding to surface
  modification and environmental condition
• . Plasmon Coupling to nearest NPs
  
• - Photoluminescence as Gold QDs
• . lt2nm of AuNPs smaller Bohr
  radius than semiconductor
• . Size dependent
  excitation/emission spectrum

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Synthesis of AuNPs

• NP Nanoparticle capped with surfactant (ex)
  sodium citrate
• MPC Monolayer-Protected Clusters with
  alkanethiol (ex) 1-OT / 11-MUA
• DEN / DSN Dendrimer-Encapsulated (or
  Stabilized) Nanoclusters

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• Schematic of a gold nanoparticle probe In
  the closed hairpin structure, the D/A pair are in
  close proximity and the fluorescence in quenched.
  Hybridization of the target single strand DNA
  opens up the structure of the molecular beacon,
  which increases the distance between the gold NP
  and the dye and results in a significant increase
  in fluorescence.

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Illustration of Surface Energy Transfer (SET)

• Schematic representation of the system,
  which consists of a fluorescein moiety (FAM)
  appended to ds-DNA of length R (varying from 15
  to 60 bp) with a Au nanoparticle (d 1.4 nm)
  appended to the other end. The flexible C6 linker
  produces a cone of uncertainty ( R) for both
  moieties. Addition of M. EcoRI (methyltransferase)
  bends the ds-DNA at the GAATTC site by 128 ,
  producing a new effective distance R'.

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• Energy transfer efficiency plotted versus
  separation distance between FAM and Au(NM).
  Filled circles () represent DNA lengths of 15bp,
  20bp, 30bp, and 60 bp. The measured efficiencies
  of these strands with the addition of M. EcoRI
  are represented by the open circles ( ). The
  error bars reflect the standard error in repeated
  measurements of the fluorescence as well as the
  systematic error related to the flexibility of
  the C6 linker as illustrated in Figure 1. The
  dashed line is the theoretical FRET efficiency,
  while the solid line is the theoretical SET
  efficiency

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Proteolytic activity monitored by FRET between
quantum dot and quencher
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• QDpeptide sensor architecture and
  optical characteristics of the fluorophores used.
  
• (a) Schematic diagram of the
  self-assembled QDpeptide nanosensors one
  peptide shown for clarity. Dye-labelled modular
  peptides containing appropriate cleavage
  sequences are self-assembled onto the QD. FRET
  from the QD to the proximal acceptors quenches
  the QD PL. Specific protease cleaves the peptide
  and alters FRET signature.
• (b) Normalized absorption and emission
  profile of dyes and QDs used Cy3 dye
  (quantum yield0.20, 150,000 M-1 cm-1, ex
  555 nm, em 570 nm), QXL-520 dark dye quencher (
  26,000 M-1 cm-1, ex 508 and 530 nm). The
  absorption of the 538 nm QDs and the emission
  spectra of the 510 and 538 nm QDs are shown.
• (C) Model structure for the QDpeptide
  conjugates. Data were derived separately but both
  conformers are shown on the same QD. The
  Casp1Cy3 peptide is shown on the right and the
  ThrQXL on the left. A CdSeZnS core-shell QD
  with a diameter of 2829 Å is represented by the
  blue inner sphere. For both peptides the His6
  sequence shown in green is in contact with the QD
  surface in an energy minimized conformation.
  Protease recognition sequences are highlighted in
  yellow, and the spacer-linker sequences are shown
  in grey. The Cy3 acceptor dye structure is shown
  in red, and the QXL-520 quencher is approximated
  by a magenta sphere placed 10.5 Å from the
  cysteine S atom. The centre-to-centre distance
  determined from FRET efficiencies are 55 Å for
  the QDCasp1Cy3 (R054 Å) and 56 Å for
  QDThrQXL (R043 Å). The second grey shell
  represents the DHLA ligand cap whose maximum
  lateral extension away from the QD surface can
  vary between 5 and 11 Å 10 Å is shown here.

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• Caspase-1 Cysteine protease
• Mediator of
  inflammation, and ssociated with apoptosis
• Thrombin Serine protease that selectively
  cleaves Arg-Gly bonds in fibrinogen
• to form fibrin and
  fibrinopeptides A and B during blood clotting
• Collagenase Metalloproteinase that specifically
  cleaves the peptide bonds in
• native triple-helical
  collagen ( Cleaves N-terminal to Gly in
  X-Gly-Pro)
• Chymotrypsin Serine protease that hydrolyzes
  peptide bonds C-terminal to
• residues
  containing aromatic or large hydrophobic side
  chains
• Collagenase and other matrix metalloproteinases
  are important pharmaceutical targets because
  they are required for cancer metastasis. Their
  aberrant expression allows tumors to invade
  healthy tissues

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Advantage vs shortcomings of FRET
Advantage

• - Relatively cheap
• - Very efficient in measuring changes in very
  proximal distances
• - Measure distances in molecules in solution
• - Only need a few µM of labeled proteins
• - Rapid detection

Shortcomings

• - Uncertainty of the orientated factor
• - When measuring a change in distance between
  two probes,
• the result is a scalar and give no
  indications of which probe
• (donor and/or acceptor) moves.
• - The presence of free labels in solution could
  mask a change in energy transfer.